Antibodies against streptococcus

ABSTRACT

Antibodies which bind the surface antigen I/II of Streptococcus sobrinus serotype d and cross react with the surface antigen I/II of Streptococcus mutans serotypes c, e, f and g.

This application is a continuation of application Ser. No. 08/481,732,filed 7 Jun. 1995, now issued as U.S. Pat. No. 5,612,031, which is acontinuation of Ser. No. 08/301,481, filed 7 Sep. 1994, now issued asU.S. Pat. No. 5,518,721, which is a continuation of Ser. No. 08/080,693,filed 22 Jun. 1993, now abandoned, which is a continuation of Ser. No.07/970,655, filed 2 Nov. 1992, now abandoned, which is a continuation ofSer. No. 07/806,814, filed 6 Dec. 1991, now abandoned, which is acontinuation of Ser. No. 07/399,515, filed 27 Oct. 1989, now abandoned,which is a 371 of PCT/GB88/00135, filed 26 Feb. 1988 published asWO88/06455, Sep. 7, 1988.

FIELD OF THE INVENTION

THIS INVENTION relates to antibodies useful to combat dental caries.

BACKGROUND TO THE INVENTION

Streptococcus mutans has been recognised for many years as the majororganism responsible for the development of dental caries in mammals.Various vaccines have been proposed in the past based on variousantigenic fragments of S. mutans. One such vaccine is described inBritish Patent No. 2,060,647 based upon the antigen known as I or I/II.Antigen I has a molecular weight, as determined by sodium dodecylsulphate polyacrylamide gel electrophoresis (SDS-PAGE) of 146-155 Kd.Antigen I/II is believed to be a conjugate of antigen I and antigen II,this I/II antigen having a molecular weight determined by SDS-PAGE of175-195 Kd. Published European Patent Application No. EP-A-0 116 472describes antigen X which is a much smaller molecule having a molecularweight, determined by SDS-PAGE of about 3.5-4.5 Kd but which appears toinclude the same antigenic determinants included within antigens I andI/II.

Antibodies against antigens I, I/II and X are known. The above-mentionedBritish Patent describes the raising of antibodies against antigens Iand I/II by conventional procedures in experimental animals, for examplerhesus monkeys, rabbits and mice. These antibodies are proposedprimarily for the purification of the antigen by affinity chromatographybut the Patent Specification mentions the possibility of using suchantibodies for passive immunisation by conventional means. Conventionalpassive immunisation involves parenteral administration of theantibodies but while such techniques are theoretically available, as apractical matter, passive immunisation has never been regarded asclinically attractive and indeed, the British Patent refers to thepreferred use of the antigenic materials for direct immunisation.

All antibodies that have been raised against S. mutans serotype c oragainst the streptococcal antigen serotype c (SA_(c)) exhibit a certaindegree of cross-reactivity. It is well known that such antibodies arealso cross-reactive with antigenic material originating from serotypes eand f of S. mutans. In clinical practice, it is found that serotypes c,e and f amount to about 90% of the bacterial S. mutans population sothat the prophylactic or therapeutic use of antibodies raised againstserotype c are of considerable practical value but fail to be effectivein relation to the residual approximately 10% of the bacterialpopulation. In some series serotype d is found in addition to serotype cin up to 50% of children examined.

This residual 10% is comprised predominantly of a serotype d that untilvery recently has been regarded merely as another serotype of S. mutans.However recently, this particular serotype has been reclassified as S.sobrinus and, in the description of this invention, we will use thenomenclature S. sobrinus serotype d rather than S. mutans serotype d.

We have now found that if antibodies are raised against S. sobrinusserotype d, that many of the resulting antibodies are cross-reactive notonly with S. sobrinus serotype d and also S. sobrinus (S. mutans)serotype g (and serotype a) but surprisingly, that such antibodies arealso cross-reactive with serotypes c, e and f of S. mutans. Sincesomewhere of the order of 98% of all serotypes of S. mutans/S. sobrinusfound in the oral cavities are of serotypes a, c, d, e, f and g, ourdiscovery has enabled us to produce, for the first time, antibodieshaving the potential for prophylactic and/or therapeutic use in relationto substantially 100% of oral bacteria of S. mutans/S.sobrinus type thatare responsible for dental caries.

Accordingly, the present invention provides antibodies against a commondeterminant of S. sobrinus serotypes d and S. mutans serotype c.

The antibodies of the present invention extend not only to wholeantibodies but also to antibody fragments containing the necessarybinding sites to enable them to recognise and bind with thestreptococcal antigen and the antibodies or fragments thereof may beprepared in polyclonal or monoclonal form.

The antibodies of the invention can be raised using S. sobrinus serotyped or streptococcal antigen derived therefrom as the immunogen. Normally,the immunogen will comprise the naturally-occurring streptococcalantigen but, as is the case with the use of streptococcal antigen fromserotype c of S. mutans, use can also be made of antigenic fragments ofserotype d, provided that those antigenic fragments contain thenecessary antigenic determinants characteristic of serotype d.

For the production of polyclonal antibodies, conventional methods may beused involving the immunisation of animals with the serotype d immunogenfollowed by recovery of the antibodies from the blood of the immunisedanimals. Conventional antibody recovery methods can be used andconventional antibody purification methods can be used, e.g. affinitychromatography using purified immunogen or fragments thereof.

Where monoclonal antibodies are to be raised, the immunogen derived fromserotype d can be used conventionally to immunise mice or other mammalsand the spleen of the immunised mammal hybridised with myeloma cells toproduce a population of hybridoma cells. Alternatively, immunogenderived from serotype d can be used for in vitro stimulation ofB-lymphocytes and the stimulated B-lymphocytes then hybridised withmyeloma cells by methods known per se to provide a population ofhybridomas. The resulting population of hybridomas may then be screenedto select those secreting monoclonal antibody that is cross-reactive byS. sobrinus serotype d and S. mutans serotype c. When polyclonalantibodies are produced, they may also be screened to select thoseshowing the serotyoe d/c cross-reactivity. The antibodies, howeverproduced, can be purified if necessary by affinity chromatographytechniques or by staphylococcal protein A to separate IgG class ofantibodies or by using serotype d antigen.

Where antibody fragments are required, the polyclonal or monoclonalantibody produced by the methods described above can be fragmented bydigestion with papain or pepsin by conventional methods.

DETAILED DESCRIPTION OF THE INVENTION

While the present invention is directed primarily to humans, the problemof dental caries is not confined to humans but arises in other mammalsincluding non-human primates, domestic and farm animals and particularlyin those cases where the non-human mammal eats a substantial proportionof food including sugars.

The monoclonal antibodies used in this invention may be applied to thetooth in the mouth of the mammal by any convenient method. Numerousmethods are now available for the treatment of teeth with variousmaterials for various purposes. If the treatment is to be carried out bya Dental Surgeon, then the monoclonal antibody is conveniently appliedby painting the surface of the tooth. If the monoclonal antibodies areto be self-applied, then the monoclonal antibodies may be included in atoothpaste, mouthwash, chewing gum, lozenge or gel. As will be describedin more detail below, the duration of protection afforded by the methodof the present invention is surprisingly long but the frequency of thetopical application is primarily one for the users personal convenience.Methods of self-application from toothpastes etc., can result inapplications being repeated perhaps daily while the use of lozenges canresult in more frequent application of the antibody. Chewing gums andgels may be regarded, for this purpose, as providing a certain amount ofsustained release of the antibody over a period of half-an-hour or moreand indeed, if sustained release of the antibody is required, thenappropriate formulations can be used that will result in slow release ofantibody into the mouth from the formulation as a result of thetemperature or saliva conditions etc., found in the mouth. In certaininstances, it may be desirable to provide a more formal prolongedcontact of the antibody with the tooth surface and in such cases,appropriate dental trays can be used that will cover the tooth after ithas been coated with antibody and prevent the antibody from beingremoved, e.g. by saliva, for a predetermined period.

It is important that the antibody be brought into contact with thesurface of the tooth and ideally should be applied to all of the smoothand occlusal surfaces of the tooth. It is not detrimental for theantibody to contact the gum but the protection afforded by the presentinvention does appear to result primarily if not exclusively from thecontact of the antibody with the surface of the tooth itself.

Topical administration of the antibodies is the most practical coursefor self-administration.

The exact amount of antibody that is applied does not appear to becritical since, in a method of this type, repeated self-application ofantibody is not difficult and indeed, particularly after initialtreatment by a Dental Surgeon, maintenance or top-up treatment can becarried out by the user at whatever frequency is desirable. By way ofguidance, it can be indicated that somewhere of the order of 10 to 100micrograms of antibody can be applied to each tooth on each occasionthat antibody is applied but amounts of antibody outside this range cancertainly be applied without causing detriment to the subject. The useof insufficient quantities of antibody simply means that the level ofprotection is not as great as would otherwise be obtainable while theuse of excessive amounts of antibody does not improve the protection andsimply results in unnecessary use of antibody.

The exact formulation for the antibody is not a matter of criticalimportance but depends entirely upon the method of application to beadopted and the convenience of the user. In all cases, it is importantto formulate the antibody in an environment of appropriate pH and whichis free from other deleterious materials which might bring about proteindegradation and the formulation should, of course, also be free frommicrobial impurity that would be deleterious in the subject's mouth. Forexample, for use in the dental surgery, the antibody could be formulatedas a simple aqueous dispersion containing somewhere in the region of 0.1to 10 milligrams of antibody per 100 microliters of liquid and a liquidof such concentration could be applied to the tooth at the rate of about1 to 10 microliters of dispersion per tooth. Where the antibody is to beformulated for self-administration, then the concentration can beselected bearing in mind the above guidelines, the quantities of theformulation that are normally taken on each occasion ofself-administration and the fact that over administration of antibodywill not be deleterious.

The following Examples are given to illustrate the invention.

MATERIALS AND METHODS Organisms

S. mutans serotypes and strains were obtained from a number oflaboratories as described previously (Smith, Lehner, Beverlev, 1984).Samples of serotype d strains (FRID and Lee) were isolated in ourlaboratories. Strain AHT, originally described as serotype a however,has been apparently reclassified to serotype g; hence no results arepresented for serotype a.

Streptococcal Antigens (SA)

SA from serotyoe d (strains FRID and OMZ 176) were prepared from culturesupernatants by combination of ammonium sulphate precipitation, ionexchange chromatography and gel filtration, as described previously(Russell et al 1980). Antigen dI/II, equivalent to cI/II of serotype c(185 Kd) was characterised using cross-reactive polyclonal rabbitantisera to SA cI/II.

Production of Monoclonal Antibodies (McAb)

McAb to SA dI/II were prepared by a method similar to that of Smith etal 1984 following the procedure of Fazekas de St. Groth and Scheidegger(1980). Briefly Balb c mice were immunised with 10 ug SA dI/TI (strainFRID) intraperitoneally in Freund's complete adjuvant, followed twoweeks later by 1.0 ug SA dI/II in Freund's incomplete adjuvant. Thethird and final injection, again two weeks later was given intravenouslyin saline and the mouse was bled and sacrificed four days later and thespleen was removed. Immune mouse spleen cells were fused at a ratio of1:1 with the mouse myeloma line P3-NS1/1-Ag4-1 (NS1), in the presence of50% (wt/vol) polyethylene glycol (PEG 4000 d; Sigma chemical Co.) inRPM1 1640 (Gibco Laboratories). The PEG was neutralised with 10N sodiumhydroxide prior to use.

After fusion the cells were plated out at 2×10⁵ total viable cells perwell, in 24 well cluster plates (Costar plastics, Cambridge, Mass.).Feeder cells from peritoneal lavage of BALB/c mice were added at 3×10⁴cell/well. Throughout cloning, RPM1 1640, supplemented with 10% fetalcalf serum, glutamine (2 mM) , pyruvate (1 mM) and antibiotics was used.For the first two weeks after fusion the medium was further supplementedwith hypoxanthine, aminopterin and thymidine to eliminate unfused NS1cells. By the third week only hypoxanthine and thymidine were added.After 10 to 14 days the supernatants of wells showing growth wereassayed for specific antibodies to the purified SA and to hearthomogenate (Bergmeier and Lehner 1983) in a micro-solid-phaseradioassay, as described previously (Smith et al 1984). Cultures givingbinding greater than 3 times the background were cloned, at least twiceby the method of limiting dilution to 0.3 cells per well and dispensedin the presence of 5×10³ feeder cells and 5×10⁴ normal spleen cells,into 96-well plates, with flat bottom wells (Costar). Cloned cells wereexpanded and then injected into pristane primed mice for ascites.Culture supernatants were stored at 4° C. with 0.1% sodium azide andascites at -20° C. The cells were frozen in the presence of 10% dimethylsulphoxide, 50% foetal calf serum in RPM1 1640 and stored in liquidnitrogen.

Characterisation of the McAb

The isotype of the McAb was determined by double diffusion precipitationin 1% agarose, using culture supernatants of the McAb and reactions withcommercial antisera (Nordic) for IgG1, IgG2a and (Eivai Bios) for IgM.The specificity of the McAb was tested by titrating each McAb againstpurified SA dI/II, derived from S. sobrinus serotype d (FRID, OMZ 176)or S. mutans serotype c (Guy's strain) as well as against whole cells ofthe other serotypes and S. sanguis (OMZ 9), by the radioassay (Smith etal 1984).

Inhibition Studies of McAb With Purified SA for Antigen Specificity

Ascitic fluids of the McAb were diluted with PBS containing 0.5% bovineserum albumin and 0.05% Tween 20, to give approximately 50% binding toSA dI/II (FRID) by micro-solid-phase radioassay. 200 μl samples ofdilute McAb were incubated with 5 μg of SA dI/II (FRID), or salineovernight at 4° C. The samples were then examined by the radioassay forantibody binding to SA dI/II.

Results

Characterisation of McAb

                  TABLE 1                                                         ______________________________________                                                  Strep. mutans  Antibody  Serotype                                   Code      antigen specificity                                                                          class     specificity                                ______________________________________                                        Guy's 1   serotype c I/III                                                                             IgG2a     c,e,f                                      Guy's 11  serotype c I/II & d I/II                                                                     IgG2b     a,c,d,e,f,g                                Guy's 12  serotype c I/II & d I/II                                                                     IgG1      c,d,e,f,g                                  Guy's 13  serotype c I/II & d I/II                                                                     IgG1      a,c,d,e,f,g                                ______________________________________                                         serotype h not tested                                                    

We have prepared and characterised various monoclonal antibodies (McAb)against various SA from serotyoe c and d (Table 1). Guy's 1 (formerly2D4), was prepared using the antigen for which it is specific asimmunogen. McAb to SA dI/II, Guy's 11, 12 and 13 were derived from asingle fusion, using SA dI/II (FRID) as immunogen. The McAb belong toIgG1, SgG2a and IgM class as shown in Table 1.

Ascitic fluid of the McAb titrate to >10⁻⁶, as measured by theradioassay against SA dI/II for Guy's 11, 12, 13 or SA cI/II forGuy's 1. Similarly, using the fluid phase radioassay against whole cellsof S. mutans, the McAb which bind to SA dI/II also bind in high titres(>10⁻⁶) to the cell surface of serotype d (OMZ 176). Guy's 1, equivalentto SA cI/II, also shows titres of >10⁻⁶ against the whole cells ofserotype c (Guy's).

SA Specificity

The specificity of McAb Guy's 1 has been demonstrated previously for SAcI/II, I, II and III by direct binding and competitive inhibition in theradioassay. Examination of the binding of Guy's 1 to SA dI/II wasnegative. However, Guy's 11, 12 and 13 react with SA I/II derived fromboth S. mutans and S. sobrinus.

Serotype Specificity

Binding of McAb to the surface of different serotypes of S. mutans andS. sobrinus was examined by the fluid phase radioassay against wholecells. Guy's 1 bound to the surface of serotype c (Guy's strain) andcross-reacted with serotype f (OMZ 175). It also showed low butsignificant binding to serotype e, giving the classical serotypecross-reactions for S. mutans (c, e, f). Guy's 11, 12 and 13 reactedwith S. mutans serotypes, c, e, f and a (except 12) and S. sobrinusserotypes d and g. None of the McAb gave any significant binding to S.sanguis.

We have prepared McAb which are specific for S. mutans serotypes c, eand f and S. sobrinus serotypes d and g. McAb to S. mutans or S.sobrinus are of the IgG1and IgG2a classes but S. sobrinus also elicitedMcAb of the IgM class. We have demonstrated the specificity of McAb forcell surface SA I/II, I, II or III by direct binding and competitiveinhibition. McAb reacting to serotype cI/II do not bind to serotypedI/II but the three monoclonal antibodies raised by immunisation with S.sobrinus showed a broad-reactivity with S. mutans and S. sobrinusserotypes. This suggests that Guy's 11, 12 and 13 are most likelydirected against a common determinant of the mutans type of streptococciand are therefore particularly convenient reagents to be used againstcolonisation of S. mutans and S. sobrinus. These SA are expressed on thecell surface (Zanders and Lehner 1981, Moro and Russell 1985) and arehere shown to bind strongly McAb in a fluid phase radioassay. The McAbshow high titres (>10⁻⁶), in binding both to purified SA and to the SAon the cell surface.

REFERENCES

1. Bergmeier L. A. and T. Lehner. 1983. Infect. Immun. 40:1075-1082.

2. Fazekas de St. Groth S. and D. Scheidegger. 1980. J. Immunol. Methods35:1-21.

3. Moro I. and M. W. Russell. 1983. Infect. Immun. 41:410-413.

4. Russell, M. W., L. A. Bergmeier, E. D. Zanders and T. Lehner. 1980.Infect. Immun. 28:486-493.

5. Russell, M. W., L. A. Bergmeier, E. D. Zanders and T. Lehner. 1980.Infect. Immun. 29:999-1006.

6. Smith, R., T. Lehner and P. C. L. Beverley. 1984. Infect. Immun.46:168-175.

7. Zanders E. D., and T. Lehner. 1981. J. Gen. Microbiol. 122:217-225.

We claim:
 1. An antibody having a binding site which binds to an epitopeof the surface antigen I/II of Streptococcus sobrinus serotype d andalso binds to the surface antigen I/II of Streptococcus mutans serotypesc, e, f and g.
 2. A binding site which binds to an epitope of thesurface antigen I/II of Streptococcus sobrinus serotype d and also bindsto the surface antigen I/II of Streptococcus mutans serotypes c, e, fand g.
 3. The binding site of claim 2 whereby said binding site wasinitially obtained using the surface antigen I/II of Streptococcussobrinus serotype d as an immunogen.
 4. The antibody of claim 1 wherebysaid binding site was initially obtained using the surface antigen I/IIof Streptococcus sobrinus serotype d as an immunogen.
 5. A binding sitewhich binds to an epitope of the surface antigen I/II of Streptococcusmutans serotypes c, e, f and g and also binds to an epitope of thesurface antigen I/II of Streptococcus sobrinus serotype d.
 6. Thebinding site of claim 5 whereby said antigen binding site was initiallyobtained using the surface antigen I/II of Streptococcus sobrinusserotype d as an immunogen.